G2/M DNA DAMAGE CHECKPOINT IN BUDDING YEAST

芽殖酵母中的 G2/M DNA 损伤检查点

• 批准号: 2022428
• 负责人: TED A. WEINERT
• 金额: $ 19.87万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022428
• 关键词: DNA binding protein; DNA damage; DNA repair; Saccharomyces cerevisiae; cell cycle; fungal genetics; gene expression; gene mutation; immunofluorescence technique; protein purification; site directed mutagenesis; temperature sensitive mutant; yeast two hybrid system

DESCRIPTION: In eukaryotic cells, DNA damage causes arrest of the cell cycle to allow repair of this damage. A number of genes, termed "checkpoint" genes, required for this arrest have been identified in yeast and in other organisms. Human checkpoint genes include p53 and ATM and mutations in these genes are associated with increased probability of developing cancer. Dr. Weinert suggests that there may be three types of checkpoint proteins: 1) sensor proteins which bind and, perhaps, process DNA damage, 2) signal proteins that interact with the sensor protein and transduce a signal to target genes, and 3) the target genes, which directly mediate cell cycle arrest. His first series of experiments concern how sensor proteins act on DNA damage. Strains with the cdc13 mutation develop long single-stranded regions near the end of the chromosome. Dr. Weinert has found that mutations in the rad24 checkpoint gene reduced this degradation, and mutations in the rad9 checkpoint gene increased the degradation. He will determine whether the degradation occurs 3' to 5' or 5' to 3', and he will examine the effects of other mutations (for example, rad17) on the degradation process. The Rad17p shares homology with Rec1p, a known 3' to 5' exonuclease. Dr. Weinert will purify Rad17p and determine whether Rad17p is a 3' to 5' exonuclease. He will also try to localize Rad17p, Rad24 and Mec3p to sites of meiosis-specific DSB's using immunofluorescence. The point of these studies is to test the hypothesis that these proteins represent sensor proteins that process the cdc13-caused DNA damage. The second part of the proposal is designed to address two questions. First, is processing of DNA damage by Rad24p and Rad17p required to repair this DNA damage? Second, does cell cycle arrest require Rad24p and Rad17p to process the DNA damage? Dr. Weinert will try to answer these questions by mutating RAD17 and RAD24, and determining whether any of the mutant alleles show separation of function. For example, if he obtains a rad17 mutant that does not process DNA damage, but does show cell-cycle arrest, he will have demonstrated that DNA processing is not required for cell-cycle arrest. The third part of the proposal concerns new checkpoint mutants. His previous hunts involved looking for mutants that had poor viability after exposure to DNA damage. His new hunt will employ a direct search for cells that fail to show G2/M arrest in response to DNA damage. He hopes that this search will identify target genes of the checkpoint response. The last series of experiments investigate the order of function of genes in the checkpoint pathway. Dr. Weinert proposes looking at protein-protein interactions between different checkpoint proteins using the two-hybrid system or affinity columns. He also proposes two types of epistasis tests. Work from the Elledge lab suggests that the Rad53p is phosphorylated as part of the checkpoint response and functions downstream of Mec1p and the sensor proteins. Dr. Weinert will attempt to confirm this conclusion. If the conclusion is confirmed, he will determine the effect of various checkpoint mutations on the phosphorylation of Rad53p. He will also attempt to isolate a mec1 mutant with the phenotype of cold-sensitive constitutive function. He will then examine interactions with other mutants. A double mutant with a mec1 constitutive and a mutant in a gene that functions downstream of MEC1 should fail to show arrest at the restrictive temperature.

描述：在真核细胞中，DNA损伤导致细胞停滞 循环，以修复这种损坏。 一些基因，被称为 在酵母中已经发现了这种抑制所需的“检查点”基因 和其他生物体。 人类检查点基因包括p53和ATM， 这些基因的突变与增加的概率有关。 发展成癌症 Weinert博士认为，可能有三种类型的 检查点蛋白：1）传感器蛋白，其结合并可能处理 DNA损伤，2）与传感器蛋白相互作用的信号蛋白， 3）靶基因，其直接 介导细胞周期停滞。 他的第一系列实验关注传感蛋白如何作用于DNA 损害 具有cdc 13突变的菌株形成长单链 靠近染色体末端的区域。 韦纳特博士发现， rad 24检查点基因的突变减少了这种降解， RAD 9检查点基因的突变增加了降解。 他将 确定降解是发生在3'到5'还是5'到3'，他将 检查其他突变（例如，rad 17）对 降解过程 Rad 17 p与Rec 1 p具有同源性，Rec 1 p是一个已知的3'端同源基因， 5'核酸外切酶。 Weinert博士将纯化Rad 17 p并确定Rad 17 p是否 是3'至5'核酸外切酶。 他还将尝试本地化Rad 17 p，Rad 24和Rad 25。 Mec 3 p与减数分裂特异性DSB位点的免疫荧光分析。 的 这些研究的目的是检验这些蛋白质 代表处理cdc 13引起的DNA损伤的传感器蛋白。 建议的第二部分旨在解决两个问题。 首先，修复需要Rad 24 p和Rad 17 p处理DNA损伤吗？ 这种DNA损伤 第二，细胞周期阻滞是否需要Rad 24 p和Rad 17 p 来处理DNA损伤 维纳特博士将尝试回答这些问题 通过突变RAD 17和RAD 24，并确定是否有任何突变体 等位基因显示功能分离。 例如，如果他获得了rad 17 突变体不处理DNA损伤，但确实显示细胞周期停滞，他 将证明细胞周期不需要DNA加工 逮捕了 提案的第三部分涉及新的检查点突变体。 他 以前的狩猎涉及寻找突变体， 暴露于DNA损伤。 他的新搜寻方法是直接搜寻细胞 其不能显示响应DNA损伤G2/M期阻滞。 他希望这一 搜索将识别检查点响应的靶基因。 最后一系列的实验研究了基因功能的顺序， 检查点路径。 Weinert博士建议研究蛋白质-蛋白质 使用双杂交技术研究不同检查点蛋白之间的相互作用 系统或关联列。 他还提出了两种类型的上位性检验。 Elledge实验室的工作表明，Rad 53 p被磷酸化， Mec 1 p和传感器的下游功能 proteins. Weinert博士将试图证实这一结论。 如果 结论得到确认后，他将确定各种检查点的效果 突变对Rad 53 p磷酸化的影响。 他还将尝试隔离 具有冷敏感组成型功能表型的mec 1突变体。 然后，他将研究与其他突变体的相互作用。 一个双重突变体， mec 1组成型和在MEC 1下游起作用的基因中的突变体 在限制性温度下不应表现出阻滞。